The present invention relates to the treatment of glomerulonephritis (GN) and other inflammatory diseases, and more generally to therapeutic treatments involving the pharmacologic inhibition of a patient""s complement system. In particular, the invention relates to the use of antibodies specific to human complement component C5 to accomplish such therapeutic treatment. The invention also relates to compositions comprising native monoclonal antibodies (mAbs) specific to human complement component C5 that block complement hemolytic activity and C5a generation at concentrations that substantially reach the theoretical one to two stoichiometric limit of antibody to antigen that can be achieved by a bivalent antibody. The invention further provides recombinant mabs that are derivatives (including monovalent derivatives) of these native mAbs that provide substantially the same blocking activities as the native mabs.
I. Immune Complex Mediated Disease
The formation of immune complexes is the typical consequence of the interaction of antigens with specific antibodies. The inflammatory response that ensues when such complexes accumulate in a limited area is an important element of normal host defenses, leading to immune complex clearance and antigen destruction by phagocytic cells. In contrast, immune complex diseases are reflections of excess complex formation or retarded clearance, usually under conditions of exceptional antigen challenge or immunologic dysregulation. Under such circumstances, immune complexes are deposited or formed at specific tissue sites and resulting inflammatory responses lead to disease states due to localized or systemic tissue damage. The kidney, and more specifically the kidney structure known as the glomerulus, is a particularly important site of immune complex deposition resulting in the development of serious disease conditions.
Human studies, and studies using animal models of human diseases, have implicated the complement system in the pathologies associated with a number of immune complex associated disorders. The activation of complement that mediates the pathology associated with these disorders may be a consequence of an autoimmune mechanism, or can be non-immunologic in origin.
The hypersensitivity response that occurs when antibodies bind to antigens either in tissues or in the circulation results from the activation of complement and the release of molecules that mediate inflammation. This process is classified as either being mediated by the binding of antibody to fixed tissue or cell bound antigens (Type II hypersensitivity) or to circulating antigens, resulting in the formation of circulating immune complexes and their subsequent pathogenic deposition in tissues (Type III hypersensitivity).
Type II hypersensitivity is mediated through the activation of complement following the binding of antibodies to fixed tissue antigens. The inflammatory response that ensues results from the activation of the proinflammatory and lytic components of the complement system and the subsequent recruitment of stimulated leukocytes to the sites of immune complex formation. The increased vascular permeability that results from the anaphylatoxic activities of C3a and C5a further enhances immune complex deposition and leukocyte recruitment.
The cross-linking of antibody bound cells or tissues to effector cells such as neutrophils, platelets, NK cells, and monocytes via their Fc receptors also plays a proinflammatory role. Such cross-linking activates effector cells, stimulating the release of oxygen radicals, prostaglandins, and leukotrienes, which release is further potentiated by the actions of activated complement components.
Examples of Type II hypersensitivity-mediated conditions include hyperacute rejection of transplanted organs, autoimmune hemolytic and thrombocytopenic states, Goodpasture""s syndrome (and associated glomerulonephritis and pulmonary hemorrhage), myasthenia gravis, pathologic sequellae associated with insulin-dependent diabetes melitus, and pemphigus vulgaris.
Type III hypersensitivity reactions involving circulating antigens can also result in the development of numerous pathologic conditions. These include glomerulonephritis (discussed in detail below), vasculitis (a potentially life-threatening inflammatory condition of large and/or small blood vessels), rheumatoid arthritis, dermatitis, and other disorders.
Other diseases associated with type III hypersensitivity reactions include autoimmune diseases such as systemic lupus erythematosis (SLE), many infectious diseases, neoplastic diseases, and a wide variety of other conditions (Dixon, et al. Immune Complex Injury, in Samter, (ed.) Immunological Diseases, 4th ed. Little Brown and Co. Boston, 1987).
II. Glomerulonephritis
The glomerulus is a key structural and functional element of the kidney. Each glomerulus is found as part of a larger structure that serves as the main functional unit of the kidney and is called a nephron. About a million nephrons are found in each kidney. Each glomerulus is a network of up to fifty parallel capillaries encased in a structure known as Bowman""s capsule. The area inside Bowman""s capsule that is not taken up by the glomerular capillaries is known as Bowman""s space. The glomerulus functions as a filter, separating water and certain solutes from the proteins and cells of the blood into Bowman""s space for further processing in the convoluted tubules, loop of Henle, and collecting duct of the nephron.
Glomerulonephritis (GN) is a disease of the kidney characterized by inflammation and resulting enlargement of the glomeruli that is typically due to immune complex formation. The accumulation of immune complexes in the glomeruli results in inflammatory responses, involving inter alia hypercellularity, that can cause total or partial blockage of the glomerulus through, among other factors, narrowing of capillary lumens. One result of this process is the inhibition of the normal filtration function of the glomerulus. Blockage may occur in large numbers of glomeruli, directly compromising kidney function and often causing the abnormal deposition of proteins in the walls of the capillaries making up the glomerulus. Such deposition can, in turn, cause damage to glomerular basement membranes. Those glomeruli that are not blocked develop increased permeability, allowing large amounts of protein to pass into the urine, a condition referred to as proteinuria.
In many cases of severe GN, pathological structures called crescents are formed within the Bowman""s space, further impeding glomerular filtration. These structures can only be seen by microscopic examination of tissue samples obtained by biopsy or necropsy, and are thus not always observed in those patients in which they occur. Crescents are a manifestation of hypercellularity and are thought to arise from the extensive abnormal proliferation of parietal epithelial cells, the cells that form the inner lining of the Bowman""s capsule. Clinical research has shown that there is a rough correlation between the percentage of glomeruli with crescents and the clinical severity of the disease, and thus the patient""s prognosis. When present in large numbers, crescents are a poor prognostic sign.
Symptoms of GN include: proteinuria; reduced glomerular filtration rate (GFR); serum electrolyte changes including azotemia (uremia, excessive blood urea nitrogenxe2x80x94BUN) and salt retention, leading to water retention resulting in hypertension and edema; hematuria and abnormal urinary sediments including red cell casts; hypoalbuminemia; hyperlipidemia; and lipiduria.
In 1990, over 210,000 patients in the United States required hemodialysis or transplantation for chronic renal failure at an annual cost in excess of 7 billion dollars, according to the United States Renal Data System (USRDS). The USRDS compiles data on kidney disease in the United States in conjunction with the National Institute of Diabetes and Digestive and Kidney Diseases, Division of Kidney, Urologic, and Hematologic Diseases, of the National Institutes of Health (NIDDKD). The USRDS estimates that the costs of treatment for renal failure are now increasing by 20 percent annually.
GN is the third leading cause of death in end-stage renal disease patients, exceeded only by diabetes and hypertension. As a result, there is a clear and long felt need in the medical community for effective treatments for this condition. Research aimed at the development of new treatments for GN is ongoing worldwide. In the United States, the NIDDKD, the National Kidney Foundation, and several other public and private organizations sponsor research in this area. The National Kidney Foundation alone supplies over two million dollars annually to fund the efforts of kidney researchers.
III. Current Treatments for GN
Corticosteroid administration, typically as high doses of xe2x80x9cpulsexe2x80x9d intravenous methylprednisolone or oral prednisone therapy, is currently considered the most effective pharmacologic agent available for the treatment of GN. Such steroid therapy is often administered in combination with cytotoxic general immunosuppressive agents such as azathioprine or cyclophosphamide. The overall immune suppression and resulting increased susceptibility to infection, along with other debilitating side effects associated with both steroid and cytotoxic drug administration, limit the effective use of these drugs.
Aspirin-like non-steroidal anti-inflammatory drugs (NSAIDs) have also been used to reduce the glomerular inflammation and enlargement of GN. These drugs are not routinely used for this purpose, however, probably because of their relatively weak anti-inflammatory effects and propensity to cause gastrointestinal and other side effects in many patients.
The administration of anticoagulants such as heparin or warfarin sodium, and antithrombotic agents such as cyproheptadine, dipyridamole, or sulfinpyrazone, has been used on the basis of evidence suggesting the involvement of the coagulation process in the genesis of glomerular crescents. However, objective evidence of benefit from such therapies in animals afflicted with experimentally induced crescentic GN has been inconsistent. Also, anticoagulants are dangerous drugs, as they can potentiate life-threatening bleeding episodes. They are especially hazardous in this regard in patients with advanced renal failure.
In addition to pharmacologic approaches, intensive plasma exchange (plasmapheresis) of 2 to 4 liters of plasma daily (or in some cases three times a week) can dramatically reduce high levels of circulating immune complexes when acute intervention in the inflammatory process is needed. Such treatment is expensive and requires that the patient be connected to the plasmapheresis machine for many hours each week. In addition, all procedures in which blood is removed from and returned to a patient are associated with an increased risk of infection. Nonetheless, plasma exchange is currently considered the most effective non-pharmacological treatment for removal of circulating immune complexes which can cause GN.
Circulating immune complex levels can also be decreased by eliminating or reducing the source of the antigen or antigens contained in the complexes by, for example, effective therapy of an underlying infection or change in an antibiotic. However, while such therapy is almost always a treatment of choice, great care must be taken since reduction of the antigen load alters the molar ratio of antigen to antibody involved in forming immune complexes and thus a dangerous temporary exacerbation of the inflammatory process may occur (see discussion below in Background Physiology and Pathology).
IV. Antibody Engineering
Native antibodies are multi-subunit animal protein molecules with highly specific antigen-binding properties. Animals make multiple classes of antibodies. There are five major classes (IgA, IgD, IgE, IgG and IgM) and a variety of subclasses. Native antibodies are made up of two or more heterodimeric subunits each containing one heavy (H) and one light (L) chain. The differences between antibody classes derive from their different H chains. H chains have a molecular weight of about 53 kDa, while L chains are about 23 kDa in mass.
Every individual native antibody has one type of L chain and one type of H chain, which are held together by disulfide bonds to form a heterodimeric subunit. Typically a native antibody (e.g., an IgG) has two such subunits, which are also held together by disulfide bonds. Within each chain, units of about 110 amino acid residues fold so as to form compact domains. Each domain is held together by a single intrachain disulfide bond. L chains have two domains, while H chains have four or five. Most H chains have a hinge region after the first (i.e., most amino-terminally located) two domains. The disulfide bonds linking together the heterodimeric subunits are located at the hinge regions. The hinge region is particularly sensitive to proteolytic cleavage, such proteolysis yielding two or three fragments (depending on the precise site of cleavage), a non-antigen binding fragment containing only H chain C regions (Fc) and one bivalent (Fabxe2x80x22) or two monovalent (Fab) antigen binding fragments. The hinge region allows the antigen binding regions (each made up of a light chain and the first two domains of a heavy chain) to move freely relative to the rest of the native antibody, which includes the remaining heavy chain domains.
The first domain of each chain is highly variable in amino acid sequence, providing the vast spectrum of antibody binding specificities found in each individual. These are known as variable heavy (VH) and variable light (VL) domains. The second and subsequent (if any) domains of each chain are relatively invariant in amino acid sequence. These are known as constant heavy (CH) and constant light (CL) domains.
Each variable region contains three loops of hypervariable sequence that provide a complementary structure to that of the antigen and are critical in determining the antigen binding specificity of the antibody, as they are the contact sites for binding to the antigen. These loops are known as complementarity determining regions, or CDRs. Each variable domain is made up of three CDRs embedded in four much less variable framework segments (FRs). Together, the sets of collinear CDRs and FRs are in large part responsible for determining the three dimensional conformation of the variable regions of antibody molecules.
CDRs and FRs are features that have been deduced from structural properties of antibody variable regions. Both amino acid sequence (primary structure) and three dimensional modeling (deduced secondary and tertiary structure) of antibody variable regions have been used by various researchers to define CDRs and, by default, FRs. While the positions of the CDRs are beyond question, not all workers in the art agree upon the precise locations of the boundaries of each CDR in VH or VL regions; there is no clear cut structural marker delineating CDR/FR boundaries.
Two definitions of CDR location are currently in general use in the art. These are the xe2x80x9csequence variabilityxe2x80x9d definition of Kabat et al. (xe2x80x9cSequences of Proteins of Immunological Interest,xe2x80x9d 4th ed. Washington, D.C.: Public Health Service, N.I.H.) and the xe2x80x9cstructural variabilityxe2x80x9d definition of Chothia and Lesk (J. Mol. Biol. 1987, 196:901). As used herein, the terms VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 refer minimally to the region of overlap between the regions designated for each CDR by each of these two definitions, and maximally to the total region spanned by the combination of the regions designated for each CDR by each of these two definitions.
One problem that antibody engineering attempts to address is the immune activity of a human patient that occurs in response to a native murine (or other non-human animal) antibody, typically a mAb, that is being administered to the patient for therapeutic purposes. This activity against murine antibodies is characterized by a human anti-mouse antibody (HAMA) response that can have deleterious effects on treatment efficacy and patient health. It has been found that almost all such human anti-non-human antibody (xe2x80x9cHAMA typexe2x80x9d) activity is directed at the constant domains and at the FR regions of the variable domains of native non-human antibodies.
By manipulating the nucleic acid molecules encoding antibody H and L chains it is possible to incorporate non-human variable regions into antibodies otherwise made up of human constant regions. The resulting antibodies are referred to as xe2x80x9cchimeric antibodies,xe2x80x9d and are typically less prone to eliciting HAMA type responses than are the non-human antibodies from which the variable regions are derived.
An even more effective approach to eliminating the potential of a non-human antibody to elicit a HAMA type response is to xe2x80x9chumanizexe2x80x9d it, i.e., to replace its non-human framework regions with human ones. One way of achieving such humanization involves the insertion of polynucleotide fragments encoding the non-human CDRs of the antibody to be humanized into a nucleic acid molecule encoding an otherwise human antibody (with human constant regions if desired) so as to replace the human CDRs and to use the resulting nucleic acid molecule to express the encoded xe2x80x9chumanizedxe2x80x9d antibody.
Unfortunately, however, humanization of non-human antibodies has unpredictable effects on antibody antigen interactions, e.g., antigen binding properties. Some of this unpredictability stems from the properties of the CDRs. Certain CDRs may be more amenable to the construction of humanized antibodies that retain the properties of the non-human CDR donor antibody than others. While the CDRs are key to the antigen binding properties of an antibody, CDRs and FRs must interact appropriately if the antigen specificity of an antibody is to be retained following humanization. The effects of combination with particular human FRs on uncharacterized non-human CDRs cannot be reliably predicted by any known method. However, the successful humanization of an antibody provides information that, in general, facilitates the successful humanization of the CDRs of that antibody using other human or altered human FRs. In addition, approaches are available that facilitate tailoring human FRs to enhance the likelihood of successful humanization.
Other problems addressed by antibody engineering include efficient antibody production and alteration of antibody pharmacokinetics. Recombinant protein production is generally most efficiently carried out in bacterial hosts. The large size and multimeric nature of native antibodies makes their production in bacteria difficult. One approach to dealing with production problems is to use recombinant DNA methods to construct antibodies that have their H and L chains joined by a linker peptide to form a single chain (sc) antibody. As described below, there are several types of sc antibodies that can be constructed.
As is the case for humanization, the effects on antigen binding properties of constructing a particular type of sc antibody using H and L chains that have not been characterized with regard to their ability to function as part of an sc antibody cannot be reliably predicted by any known method. However, the successful construction of any one type of sc antibody from a particular native antibody provides information that, in general, facilitates the successful construction of other types of sc antibodies from that native antibody.
Single chain antibodies may include one each of only VH and VL domains, in which case they are referred to as scFv antibodies; they may include only one each of VH, VL, CH, and CL domains, in which case they are referred to as scFab antibodies; or they may contain all of the variable and constant regions of a native antibody, in which case they are referred to as full length sc antibodies.
The differing sizes of these antibodies imparts each with differing pharmacokinetic properties. In general, smaller proteins are cleared from the circulation more rapidly than larger proteins of the same general composition. Thus, full length sc antibodies and native antibodies generally have the longest duration of action, scFab antibodies have shorter durations of action, and scFv antibodies have even shorter durations of action. Of course, depending upon the illness being treated, longer or shorter acting therapeutic agents may be desired. For example, therapeutic agents for use in the prevention of immune and hemostatic disorders associated with extracorporeal circulation procedures (which are typically of brief duration) are preferably relatively short acting, while antibodies for the treatment of long term chronic conditions (such as inflammatory joint disease or GN) are preferably relatively long acting.
Detailed discussions of antibody engineering may be found in numerous recent publications including: Borrebaek, xe2x80x9cAntibody Engineering, A Practical Guide,xe2x80x9d 1992, W.H. Freeman and Co. NY; and Borrebaek, xe2x80x9cAntibody Engineering,xe2x80x9d 2nd ed. 1995, Oxford University Press, NY, Oxford.
In view of the foregoing, it is an object of the present invention to provide a new approach for reducing the glomerular inflammation and kidney dysfunction associated with GN.
The method of the invention involves the use of preparations containing antibodies to human complement component C5 as pharmaceutical agents. More particularly, the invention provides for the use of anti-C5 antibodies that bind to complement component C5 or active fragments thereof. Preferably, the antibodies block the generation and/or activity of complement components C5a and C5b. For most applications, the antibody is a monoclonal antibody.
In the preferred embodiments of the invention, the administration of the anti-C5 antibody preparation is started after the appearance of GN symptoms, e.g., after the appearance of proteinuria. Alternatively, the invention can be used prophylactically to treat patients who are at risk for an acute exacerbation of existing GN, e.g., patients experiencing a flare-up of symptoms of systemic lupus erythematosus or similar autoimmune diseases that have resulted in GN.
As shown in the examples presented below, anti-C5 antibodies administered subsequent to the onset of GN essentially eliminate glomerular inflammation/enlargement and reduce kidney dysfunction (see Examples 1 and 2).
Although not wishing to be bound by any particular theory of operation, it is believed that the anti-C5 antibodies have these and other therapeutic effects through their activity in blocking the generation or activity of the C5a and/or C5b active fragments of complement component C5. Through this blocking effect, the antibodies inhibit the proinflammatory (anaphylatoxic) effects of C5a and the generation of the C5b-9 membrane attack complex (MAC). Significantly, the blockage effected by the anti-C5 antibodies, since it occurs at the level of complement component C5, has the advantage of maintaining important opsonic, anti-infective, and immune complex clearance functions of the complement system mediated by, inter alia, complement component C3.
The invention additionally provides compositions comprising anti-C5 antibodies that block complement hemolytic activity and C5a generation. These antibodies are useful for the treatment of GN as well as a number of other conditions. These include treatment of immune and hemostatic dysfunctions associated with extracorporeal circulation (see copending U.S. patent application Ser. No. 08/217,391, now U.S. Pat. No. 5,853,722 which is incorporated herein by reference), treatment of inflammatory joint diseases (see copending U.S. patent application Ser. No. 08/311,489, which is incorporated herein by reference), and other complement associated conditions, particularly inflammatory diseases.
Although other antibodies can be used to treat GN in accordance with the present invention, the novel antibodies of the invention are preferred. Preferably, these novel antibodies bind to the alpha chain of C5, but do not exhibit substantial binding to the alpha chain cleavage product C5a (referred to hereinafter and in the claims as xe2x80x9cfree C5axe2x80x9d). Other preferred targets for antibody binding include fragments of the alpha chain of human C5 that are immunoreactive with the most preferred antibody of the invention, the 5G1.1 antibody discussed below. Such preferred targets include the 46 kDa acid hydrolysis fragment of C5 (the xe2x80x9c5G46kxe2x80x9d fragment), the 27 kDa tryptic digestion fragment of C5 (the xe2x80x9c5G27kxe2x80x9d fragment), the 325aa peptide spanning amino acid residues 725-1049 of SEQ ID NO:2 (the xe2x80x9c5G325aaxe2x80x9d peptide), the 200 amino acid peptide spanning amino acids residues 850 to 1049 of SEQ ID NO:2 (the xe2x80x9c5G200aaxe2x80x9d peptide)xe2x80x94as discussed below in Example 13.
The novel antibodies of the invention include antibodies that bind to an epitope within the amino acid sequence Val Ile Asp His Gln Gly Thr Lys Ser Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser, (SEQ ID NO:1) hereinafter referred to as the KSSKC epitope. These novel antibodies that bind to the KSSKC epitope (SEQ ID NO:1) are hereinafter referred to as anti-KSSKC antibodies, and monoclonal antibodies binding to the KSSKC epitope are hereinafter referred to as anti-KSSKC mAbs.
The novel antibodies of the invention have many advantages over other anti-C5 antibodies, particularly with regard for their use as anti-inflammatory therapeutic agents. These include the ability to substantially block both complement hemolytic activity and the generation of the proinflammatory complement cleavage product C5a to substantially the same extent at the same concentration of antibody. Some of the preferred antibodies of the invention have the additional advantageous property of blocking the binding of C5 to C3 or C4.
Particularly preferred antibodies of the invention are monospecific native anti-KSSKC antibodies. The 5G1.1 native anti-KSSKC mAb has the distinct advantage of substantially blocking both complement hemolytic activity and the generation of C5a at a stoichiometric ratio of antibody to C5 that approaches the theoretical one to two (antibody to antigen) limit of binding that can be achieved by a bivalent antibody. This is a desirable property because it allows smaller doses of antibody to achieve therapeutic effects than would be required of otherwise similar antibodies that cannot function at such a ratio.
The invention further provides recombinant mAbs that are derivatives (including monovalent derivatives) of these native mAbs. These include anti-KSSKC recombinant mabs. Preferably the antibodies of the invention provide a level of blockade of both complement hemolytic activity and C5a generation (on a per mole of binding site basis) that is obtained when the antibody concentration is within an order of magnitude of that of the native mAbs. Particularly preferred anti-KSSKC recombinant mAbs provide a level of such blockade when the antibody concentration is no more than three fold that of the native mAbs of the invention.
The invention further provides nucleic acid sequences of polynucleotides encoding such recombinant anti-KSSKC mAbs, as well as amino acid sequences of the polypeptides encoded by these nucleic acid molecules of the invention.
The invention further provides CDR sequences that are useful in the construction of the humanized antibodies of the invention, as well as peptides and oligopeptides that are useful in the preparation and characterization of the antibodies of the invention.
Anti-C5 antibodies of the invention have activity in blocking the generation or activity of the C5a and/or C5b active fragments of complement component C5. Through this blocking effect, the antibodies inhibit the proinflammatory (anaphylatoxic) effects of C5a and the generation of the C5b-9 membrane attack complex (MAC). Significantly, the blockage effected by the anti-C5 antibodies, since it occurs at the level of complement component C5, has the advantage of maintaining important opsonic, anti-infective, and immune complex clearance functions of the complement system mediated by, inter alia, complement component C3.
The accompanying figures, which are incorporated in and constitute part of the specification, illustrate certain aspects of the invention, and together with the description, serve to explain the principles of the invention. It is to be understood, of course, that both the figures and the description are explanatory only and are not restrictive of the invention.